1 Sex makes them sleepy: change in host reproductive status induces diapause in 2 parasitoidsa population experiencing harsh winters

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6 Tougeron K.1, 2, Brodeur J.2, van Baaren J.1, Renault D.1, 3 & Le Lann C.1

7 1 Univ Rennes, CNRS, ECOBIO (Ecosystèmes, biodiversité, évolution) - UMR 6553, 263 Avenue du 8 Général Leclerc, F-35000 Rennes, France.

9 2 Institut de Recherche en Biologie Végétale, Département de Sciences Biologiques, Université de 10 Montréal, 4101 rue Sherbrooke Est, Montréal, QC, Canada, H1X 2B2.

11 3 Institut Universitaire de France, 1 rue Descartes, 75231 Paris Cedex 05, France

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14 Corresponding author: [email protected]

15 Current address: The University of Wisconsin – La Crosse, Department of Biology, La Crosse, 16 Wisconsin, United States of America, 1725 State street, 54601

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21 Abstract

22 When organisms coevolve, any change in one species can induce phenotypic changes in 23 traits and ecology of the other species. The role such interactions play in ecosystems is central, 24 but their mechanistic bases remain underexplored. Upper trophic level species have to 25 synchronize their life-cycle to both abiotic conditions and to lower trophic level species’ 26 phenology and phenotypic variations. We tested the effect of host seasonal strategy on 27 parasitoid diapause induction by using a holocyclic clone of the pea aphid Acyrthosiphon pisum 28 producing two morphs with either asexual (and sexual morphs that are viviparous females) or 29 sexual ( (i.e. laying embryos) and oviparous females) reproduction (laying eggs), respectively, 30 the latter being only present at the end of the growing season. ervi from 31 populations of contrasted climatic origin (harsh vs. mild winter areas) were allowed to parasitize 32 each morph in a split-brood design and developing parasitoids were next reared under either 33 fall-like or summer-like temperature-photoperiod conditions. We next examined aspects of the 34 host physiological state by comparing the relative proportion of forty-seven metabolites and 35 lipid reserves in both morphs. produced under the same conditions. We found that oviparous 36 morphs are cues per se for diapause induction; parasitoids entered diapause at higher levels 37 when developing in oviparous hosts (19.4 ± 3.0 %) than in viviparous ones (3.6 ± 1.3 %), under 38 summer-like conditions (i.e.., when oviparous aphids appear in the fields). This pattern was only 39 observed in parasitoids from the harsh winter area since low diapause levels were observed in 40 the other population-dependent, suggesting local adaptations to overwintering 41 cues. Metabolomics analyses show parasitoids’ response to be mainly influenced by the host’s 42 physiology, with higher proportion of polyols and sugars, and more fat reserves being found in 43 oviparous morphs. Host quality thus varies across the seasons. and represents one of the 44 multiple environmental parameters affecting parasitoid diapause. Our results underline strong 45 coevolutionary processes between hosts and parasitoids in their area of origin, likely leading to 46 phenological synchronization, and we point out theirthe importance of such bottom-up effects 47 for trait-expression, and for the provision of ecosystem serviceservices such as biological 48 control in the context of climate change.

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50 Key-words

51 Coevolution; Phenotypic plasticity; Phenology; Host-parasite synchronization; 52 Environmental cue; Metabolomics

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57 Introduction

58 Interacting individuals from two biological entities can adjust their phenotypes in response to 59 cues from each other, even when these cues vary across time (Agrawal 2001). Beneficial or 60 antagonistic interactions, from mutualism to parasitism, predation and competition may lead to 61 adaptive phenotypic responses. When interactions persist over generations, coevolution can 62 occur and species adapt to the interacting species’ life history traits, phenology and ecology 63 (Agrawal 2001, Ellers et al. 2012). Interaction norms (Thompson 1988) arise from ecological 64 responses of interacting organisms in varying environments, as any phenotypic change 65 occurring in one ―partner‖ species can cascade to the other species’ phenotype (Fordyce 2006, 66 Hughes 2012). Cues produced by one interacting species may indirectly inform the other species 67 of environmental changes. For example, plant senescence in fall can inform herbivorous 68 of upcoming detrimental winter conditions and induces phenotypic changes (e.g. diapause 69 induction) or migration behaviour (Archetti et al. 2009).

70 Parasitoids are excellent models to study phenotypic expression in interacting species 71 because they are strongly influenced during immature stages by changes in nutritional and 72 physiological quality of their host (Godfray 1994). Diapause is an important ecological process 73 in insects allowing them to survive recurrent unfavorable environmental conditions (Tauber et 74 al. 1986). For parasitoids, diapause also contributes to maintain synchronization with their 75 host’s seasonal reproductive-cycle; it is induced before suitable hosts vanish from the 76 environment (Lalonde 2004). As in most insects, diapause in parasitoids is mainly induced by 77 abiotic cues perceived either by the generation that will enter diapause, or by the maternal 78 generation (Tauber et al. 1986). A few studies also reported that diapause in parasitoids can be 79 triggered by the onset of host diapause (Polgár and Hardie 2000, Gerling et al. 2009), or through 80 intraspecific competition for hosts (Tougeron et al. 2017a). However, whether the phenotype of 81 a non-diapausing host can influence parasitoid diapause remains poorly studied.

82 Aphids are hosts for parasitoids and can have very complex cycles showing 83 seasonal alternation between morphs with asexual and sexual reproduction (Dixon 1985)(Dixon 84 1985). Asexual females reproduce parthenogenetically and lay live offspring (i.e. viviparity) 85 whereas sexualsexually reproducing females produce eggs (i.e. oviparity) after mating with 86 males. Sexual aphid morphs are present at higher proportions in harsh than in mild winter 87 climates (Dedryver et al. 2001)(Dedryver et al. 2001), and they represent the last hosts available 88 for aphid parasitoids before winter as they produce overwintering eggs in fall (Leather 89 1992)(Leather 1992). Consequently, sexual morphs have been suggested to promote diapause in 90 parasitoids, indicating a host physiological effect (Polgár et al. 1991, 1995, Christiansen- 91 Weniger and Hardie 1997)(Polgár et al. 1991, 1995, Christiansen-Weniger and Hardie 1997). 92 No mechanistic understanding of this phenomenon has been proposed and the effects of the host 93 morph have not been detangled from confounding factors such as host genotype and geographic 94 origin, host size, abiotic conditions, or the season at which hosts are sampled in the fields. Hosts 95 and parasitoids share common evolutionary historyhave coevolved over long periods of time, 96 they respond to similar seasonal cues and the physiological syndrome associated with 97 overwintering is highly conserved among insects (Tauber et al. 1986, Denlinger 2002)(Tauber et 98 al. 1986, Denlinger 2002). As a result, the related physiological state of the host may represent a 99 reliable signal of upcoming seasonal changes for parasitoids.

100 Hormones, fats, carbohydrates and other types of metabolites are involved in the control of 101 overwintering and diapause expression in insects (Chippendale 1977, Christiansen-Weniger and

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102 Hardie 1999, Denlinger 2002, Sinclair and Marshall 2018). In aphid parasitoids, metabolomic 103 and proteomic profiles differ between diapausing and non-diapausing individuals, with higher 104 amounts of sugars, polyols and heat shock proteins being found in diapausing parasitoids 105 (Colinet et al. 2012). In aphids, morphs differ in morphology and physiology; oviparous females 106 accumulate reserves to produce energetically costly diapausing eggs (Le Trionnaire et al. 2008) 107 with cryoprotectant compounds such as mannitol and glycerol (Sömme 1969), whereas 108 viviparous females metabolize energetic resources rapidly to produce embryos. Aphids’ 109 triglyceride reserves change quantitatively and qualitatively across the seasons with alternating 110 morphs (Greenway et al. 1974). Immature parasitoids are known to consume sugars and lipids 111 from their hosts (Jervis et al. 2008) and are therefore influenced by host reserves for their 112 growth and development.

113 We questioned the extent to which oviparous and viviparous morphs of a single clone of the 114 pea aphid Acyrthosiphon pisum (Harris) (Hemiptera: Aphididae) influences winter diapause 115 expression in the parasitoid Aphidius ervi Haliday (: ) under summer 116 and fall conditions. Under laboratory conditionsHormones, fats, carbohydrates and other types 117 of metabolites are involved in the regulation of overwintering and diapause expression in insects 118 (Chippendale 1977, Christiansen-Weniger and Hardie 1999, Denlinger 2002, Sinclair and 119 Marshall 2018). In aphid parasitoids, metabolomic and proteomic profiles differ between 120 diapausing and non-diapausing individuals, with higher amounts of sugars, polyols and heat 121 shock proteins being found in diapausing parasitoids (Colinet et al. 2012). In aphids, morphs 122 differ in morphology and physiology; oviparous females accumulate reserves to produce 123 energetically costly diapausing eggs (Le Trionnaire et al. 2008) with cryoprotectant compounds 124 such as mannitol and glycerol (Sömme 1969), whereas viviparous females metabolize energetic 125 resources rapidly to produce embryos. Aphids’ triglyceride reserves change quantitatively and 126 qualitatively across the seasons with alternating morphs (Greenway et al. 1974). Immature 127 parasitoids are known to consume sugars and lipids from their hosts (Jervis et al. 2008) and are 128 therefore influenced by host reserves for their growth and development.

129 We questioned the extent to which oviparous and viviparous morphs of a single clone of the 130 pea aphid Acyrthosiphon pisum (Harris) (Hemiptera: Aphididae) influences winter diapause 131 expression in the parasitoid Aphidius ervi Haliday (Hymenoptera: Braconidae) under summer 132 and fall conditions. Under laboratory conditions and using a split-brood design, we compared 133 the response to two aphid morphs of two populations of parasitoids from mild (France) and 134 harsh (Canada) winter areas that differed in their level of diapause expression (Tougeron et al. 135 2018)(Tougeron et al. 2018). In Aphidius species, winter diapause is initiated at the prepupal 136 stage within the aphid mummy (i.e. dead aphid containing a developing parasitoid) following 137 stimuli perceived by the mother or early developmental stages (Brodeur and McNeil 1989, 138 Tougeron et al. 2017b)(Brodeur and McNeil 1989, Tougeron et al. 2017b). We hypothesized 139 that parasitoids of both populations developing in oviparous hosts enter diapause at higher 140 proportions than those developing in viviparous hosts, independently of photoperiod and 141 temperature. We predicted this pattern to originate from differences in aphids’ physiological 142 contents. We thus performed physiological analyzes to measure lipid content and quantify aphid 143 morphs metabolites. We also hypothesized parasitoids from mild winter area to be less 144 responsive to diapause inducing cues from the host and the environment, because parasitoid 145 populations should be adapted to climatic conditions and relative occurrence of sexual hosts in 146 their respective area of origin.

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148 Material and Methods

149 Biological materials

150 Two populations of the parasitoid A. ervi were collected in 2015 at the mummy stage in pea 151 fields from two contrasted climatic origins: near Montréal, QC, Canada (45.584°N, 73.243°W; 152 harsh winter area) and near Rennes, France (48.113°N, 1.674°W; mild winter area). Parasitoids 153 were then reared under controlled conditions using a cyclically parthenogenetic clone (clone F2- 154 X9-47) of the pea aphid A. pisum provided by INRA Le Rheu, France, and known to produce 155 both oviparous and viviparous aphid morphs (Jaquiéry et al. 2014).One population per 156 geographic origins was used as high gene flow has been reported in A. ervi populations which 157 therefore present little genetic differentiation (Hufbauer et al. 2004). Even if gene flow was 158 weak, we would expect higher differences between Canadian and French populations than 159 among populations of a same location. Parasitoids were then reared under controlled conditions 160 using a cyclically parthenogenetic clone (clone F2-X9-47) of the pea aphid A. pisum provided 161 by INRA Le Rheu, France, and known to produce both oviparous and viviparous aphid morphs 162 (Jaquiéry et al. 2014). The symbiotic load of the aphid clone we used was not assessed, but 163 symbionts present in the grandparent generation from which our clone comes from had been 164 identified. Half of the grandparent was associated with Serratia symbiotica, the other half had 165 no secondary endosymbionts (J. Jaquiéry pers. comm.), it is thus likely that our clone was 166 inhabited by S. symbiotica. All insects were maintained on fava bean Vicia faba (Fabaceae) at 167 20 °C, 70% relative humidity (RH) and 16:8 h Light:Dark (L:D) photoregime.

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169 Production of sexual and asexual hosts

170 Three aphid morphs were used in the experiments; oviparous females (O), viviparous 171 females (V) and a control treatment for viviparous females (C), as detailed below.

172 Three parthenogenetic A. pisum adult females from the aphid culture were put on bean plants 173 (N=15) and allowed to lay larvae during four days at 20 °C, 70% RH, 16:8 h LD.L:D. Females 174 were then removed and infested plants were put in a growing chamber at 17 °C, 70% RH, 12:12 175 h (L:D), and under 36W, IRC 85, 6500 K day-light type fluorescent tubes to induce the 176 production of sexual aphids (Le Trionnaire et al. 2009).(Le Trionnaire et al. 2009). At each 177 generation, plants were renewed, and less than five aphids were maintained per plant to prevent 178 formation of alatewinged individuals due to overcrowding (Hardie 1980).(Hardie 1980). As 179 embryos directly detect photoperiodic cue through the cuticle of the grand-mother (Le 180 Trionnaire et al. 2008)(Le Trionnaire et al. 2008), the first sexual aphids: males (~20%) and 181 oviparous females (30 to 60%) were formed, along with asexual aphids (20 to 50%): sexuparous 182 (a particular type of parthenogenetic females producing sexual morphs) and viviparous aphids 183 (parthenogenetic females producing only parthenogenetic morphs), after three generations under 184 these conditions. As sexuparous and viviparous aphids cannot be distinguished 185 morphologically, they were indistinctly considered as the ―viviparous female‖ treatment. 186 However, a control group of viviparous parthenogenetic females (C) was produced by rearing 187 aphids under non-sexual-inductive conditions (20 °C, 70% RH, 16:8 h L:D). This treatment 188 controls for potential stress effects of the sexual-inductive conditions on the aphid, and allows to 189 solely measuring the response of viviparous aphids as sexuparous are not produced under this 190 condition (Dixon 1985)(Dixon 1985). Oviparous aphid morphs were differentiated from 191 viviparous ones under a stereo microscope (x10) by observing the morphology of their legs:

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192 oviparous female aphids have rhinaria on the tibia, and have a femur of the same width as the 193 tibia, and viviparous females have a wider tibia than the femur without rhinaria (Lamb and 194 Pointing 1972, Hullé et al. 2006)(Lamb and Pointing 1972, Hullé et al. 2006). Aphid males 195 were not included in our analyses since A. ervi does not parasitize them, probably because they 196 are too small and have lower energetic reserves than female morphs (Tougeron et al., 197 unpublished data).

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199 Diapause induction

200 Aphid mummies from the colonies were isolated in a small gelatin capsule until parasitoid 201 emergence. Newly emerged parasitoids were put in a 5 cm plastic tube for mating (5 females 202 with 2 males) for 24 h, and were fed with a 70 % diluted honey solution. Maternal genotype, 203 egg-laying order in different aphid morphs, in addition to parasitoids’ age or host preference 204 may affect diapause induction (Brodeur and McNeil 1989)(Brodeur and McNeil 1989). To 205 consider these potential effects, twelve A. ervi females were individually allowed to parasitize 206 16 adult aphids of the same age and size within the same cohort and of each of the three morph 207 types (oviparous female, viviparous female, control viviparous females produced under non- 208 sexual-inductive conditions, N=48 aphids offered for parasitism per female wasp) for 12 h over 209 three consecutive days, by alternating the order of presentation of aphid morphs among females. 210 Parasitoids rested at night, with an access to diluted honey. Aphids were introduced in a plastic 211 tube (10 x 3 cm) and were given a few minutes to settle on a bean cut plant, after which a 212 parasitoid was introduced into the tube. Four parasitoid females were first individually put in 213 presence of oviparous aphids, then moved to a second tube with control viviparous aphids and 214 next moved to a third tube containing viviparous aphids (OCV). Four other females were first 215 offered viviparous aphids (VOC), and the last four females were first offered control viviparous 216 aphids (CVO) (Fig. 1).

217 After each oviposition period, the 16 potentially parasitized aphids of each morph type were 218 transferred by group of 8 on two bean plants. Plants were next enclosed into micro-perfored 219 plastic bags and placed at either 20 °C, 16:8 h (L:D) (summer-like conditions not inducing 220 diapause in A. ervi) or 17 °C, 10:14 h (L:D) (autumn-like conditions inducing diapause) 221 (Tougeron et al. 2017b)(Tougeron et al. 2017b). When the plants began to wilt, aphids were 222 transferred to another plant with a small paintbrush. Mummification was checked daily, and 223 newly-formed mummies were placed individually into gelatin capsules, and remained under 224 their respective temperature and photoperiod treatments until adult emergence. Mummies from 225 which no parasitoid had emerged 15 days after mummification were dissected, and the content 226 was recorded as dead parasitoids or diapausing individuals (golden-yellow prepupae, Tougeron 227 et al. 2017b)Tougeron et al. 2017b). This experiment was repeated twice per parasitoid 228 population; diapause levels were thus calculated among the offspring of 24 females for each 229 treatment. Patterns were consistent in each of the repeated experiments. Our split-brood family 230 design also allowed comparing reaction norms (RN) of diapause levels in the offspring of each 231 parasitoid female from each population, both within morphs at different abiotic conditions, and 232 within abiotic conditions among morphs. We have excluded ―control‖ morphs from the RN 233 analysis as their effect on diapause induction did not differ from viviparous morphs.

234 The aphid morph (individual differences within a population due to developmental plasticity) 235 and the aphid clone (differences in reproduction modes genetically determined between

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236 populations) may both influence parasitoid diapause. To consider this aspect, we compared the 237 incidence of diapause when parasitoids developed in the cyclically parthenogenetic clone 238 (holocyclic, i.e., alternating between sexual and sexual morphs) described above and in an 239 obligate parthenogenetic clone, producing only viviparous females (anholocyclic clone F2-X9- 240 19; Jaquiéry et al. 2014)Jaquiéry et al. 2014). To achieve this goal, five A. ervi females were 241 individually allowed to sequentially parasitize 35 viviparous aphids of each clone during 12 h. 242 Parasitized hosts were next placed at 17 °C 10:14 h (L:D), and diapause induction was 243 measured as described above. We excluded any clone effect because diapause incidence was 244 similar for parasitoids developing in viviparous aphids of either the holocyclic (59.9 ± 10.1 %, 245 n=132 mummies) or the anholocyclic (66.0 ± 7.7 %, n=112 mummies) clone (GLM, p=0.97). 246 The cyclically parthenogenetic clone was thus used for the experiments.

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250 Figure 1: Experimental design for diapause induction in the parasitoid Aphidius ervi. Twelve parasitoid 251 females were individually allowed to parasitize 16 Acyrthosiphon pisum from each of the three host 252 morphs for 12 h: oviparous (O), viviparous (V) and viviparous control (C). First contact (parasitism 253 sequence) with an aphid was alternated between the three morphs (OCV, VOC, CVO). Following 254 parasitism, the aphid cohort was split in two and individuals were reared under a diapause-inductive 255 condition (17 °C 10:14 h L:D) or a non-diapause-inductive condition (20 °C 16:8 h L:D). This protocol 256 was repeated twice for parasitoid populations originating from mild or harsh winter.

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258 Metabolomic analyses and lipid reserves

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259 NonAs sexual morphs could only be produced at 17°C, we compared non-parasitized 260 apterous adult aphids of viviparous and oviparous females of the same age (between 24 and 48 h 261 after imago molt), produced under the same conditions used for the diapause experiment (at 17 262 °C, 12:12 h (L:D)),)). Samples were kept at -20 °C for metabolomic and lipid analyses. 263 SamplesThey were dried out at 60 °C for 2 days in a freeze-dryer and their dry mass measured 264 using a Mettler-Toledo precision balancescale (accurate to 0.001 mg). Viviparous aphids’ dry 265 mass ranged from 0.280 mg to 0.742 mg, and oviparous aphids’ dry mass ranged from 0.358 mg 266 to 0.739 mg.

267 For metabolic analyses, 18 aphids of each morph (viviparous and oviparous females) were 268 used. Nine replicates were analyzed for each morph condition, each consisting of a pool of two 269 aphid females. The samples were put in 600 µL of chlorophormchloroform-methanol (1:2) 270 solution and homogeneizedhomogenized using a tungsten-bead beating apparatus at 30 Hz for 271 1.5 min. Then, 400 µL of ultrapure water was added to each tube and samples were centrifuged 272 at 4 °C, 4,000 g for 5 min. Finally, 90 µL of the upper aqueous phase containing metabolites 273 were transferred to chromatographic vials. Injection order of the samples was randomized prior 274 mass spectrometry detection. Metabolomic fingerprinting process was performed following the 275 protocol of Khodayari et al. (2013)Khodayari et al. (2013). Chromatograms were analyzed 276 using XCalibur software (Thermo Fischer Scientific, Waltham, MA, USA). We accurately 277 quantified 47 metabolites: 14 amino acids, 11 sugars / sugar phosphates, 8 organic acids, 7 278 polyols, 4 other metabolites and 3 amines (Table 1). Details of metabolite amounts measured 279 from each morph are provided in Figure S1.

280 Lipid contents were measured using 52 oviparous females and 23 viviparous females. Each 281 dry aphid was left for two weeks in a microtube containing 1 mL of chloroform-methanol 282 solution (2:1) to extract lipids (Terblanche et al. 2004)(Terblanche et al. 2004). Aphids were 283 then rinsed with the same solution, and placed back in the freeze-dryer for 24 h to eliminate the 284 residues of the extracting solution and next weighted again to measure fat content (= fat mass 285 (mg) / lean dry mass (mg), Colinet et al. 2007). Code de champ modifié

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287 Table 1: Metabolites detected in each of the two morphs (viviparous and 288 oviparous females) of the pea aphid, Acyrthosiphon pisum. Each metabolite has 289 been found in each morph. Abbreviations used on Figure 13 are in brackets. Amino acids Organic acids Alanine (Ala) Citric acid (Cit_Ac) Aspartic acid (Asp_Ac) Galacturonic acid (Gal_Ac) Citrulline (Citr) Glyceric acid (Glyc_Ac) Glutamic acid (Glu) Lactic acid (Lact_Ac) Glycine (Gly) Malic acid (Mal_Ac) Isoleucine (Ile) Phosphoric acid (Phos_Ac) Leucine (Leu) Pipecolic acid (Pipe_Ac) Lysine (Lys) Quinic acid (Quin_Ac) Ornithine (Orn) Sugars and sugar phosphates Proline (Pro) Arabinose Serine (Ser) Fructose Valine (Val) Fructose-6-phosphate (F6P) Threonine (Thr) Galactose Phenylalanine (Phe) Glucose Polyols Glucose-6-phosphate (G6P) Adonitol Maltose Arabitol Mannose Galacticol Ribose Glycerol Saccharose

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Inositol Trehalose Mannitol Other metabolites Xylitol Gluconolactone (GNL) Amines Gamma aminobutyric acid (GABA) Cadaverine (Cad) Glycerol-3-phosphate (Gly3P) Triethanolamine (TEA) Dopamine (Dop) Putrescine (Put) 290

291 Statistical analyses

292 Generalized linear mixed-effects models (GLMM) with binomial distributions were fit to the 293 data using the lme4 package. The response variable was the proportion of diapausing 294 parasitoids; the origin of the parasitoid population (Canada vs. France), the host morph (three 295 modalities, O, V, C), the temperature/photoperiod conditions (17°C 10:14h vs 20°C 16:8h), and 296 their interaction, were considered as fixed factors; the identity of each parasitoid female and the 297 egg-laying (parasitism) order were considered as random effect factors in the models. As 298 diapause incidence differed between parasitoid populations (GLMM, χ²=216, df=1, p<0.001), 299 data from both populations were analyzed separately using similar GLMMs. Significance of 300 each term in the model was analyzed using the package car.

301 For metabolite data, concentrations of the compounds were first log-transformed. Then, a 302 Principal Component Analysis (PCA) was performed to detect which metabolites (expressed in 303 nmol.mg-1) differed the most between host morphs. Log-transformed metabolite concentrations 304 were then summed up among each category (Table 1) and another PCA was performed using 305 metabolite groups as discriminatory factors. An ANOVA with FDR-adjusted p-values was next 306 performed to compare concentrations of each metabolite between morphs. Finally, an ANOVA 307 tested differences in fat content between oviparous and viviparous morphs. All statistical 308 analyses were carried out using the R software (R Core Team 2017)(R Core Team 2017).

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310 Results

311 Diapause incidence in the parasitoid A. ervi

312 HostIn the Canadian (harsh winter area) population, diapause levels were affected by host 313 morph (GLMM, χ²=12.6, df=2, p<0.001; Fig. 2) and abiotic conditions (GLMM, χ²=250.0, 314 df=1, p<0.001), with an interaction effect as host morphs influenced parasitoid diapause 315 incidence only in the Canadian (harsh winter area) population at 20 °C 16:8 h (L:D) (GLMM, 316 χ²=16.9, df=2, p<0.001; Fig. 2). Diapause incidence was higher at 17 °C 10:14 h L:D than at 20 317 °C, 16:8 h L:D, for the Canadian population (76.9 ± 2.5% vs. 9.0 ± 1.5%, respectively). At 20 318 °C, 16:8 h L:D, diapause incidence was higher when Canadian parasitoids developed in 319 oviparous aphids (19.4 ± 3.0 %)% s.e.) than in viviparous aphids (3.6 ± 1.3 %)%, z=-4.3, 320 p<0.001) or viviparous control aphids (3.8 ± 1.4 %). The%, z=-3.9, p<0.001).

321 In the French (mild winter area) population, the host morph did not have an effect on 322 diapause at 17 °C 10:14 h LD for both influence parasitoid populations (Fig. 2). diapause 323 (GLMM, χ²=1.84, df=2, p=0.39), abiotic conditions did influence parasitoid diapause (GLMM, 324 χ²=237.9, df=1, p<0.001), but no interaction effect can be interpreted since no diapause was 325 expressed for the French population at 20 °C, 16:8 h L:D. Diapause incidence was higher at

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326 17 °C 10:14 h L:D than at 20 °C, 16:8 h L:D, for the French population (27.9 ± 2.1% vs. 0%, 327 respectively). Random factors female identity and host exposition order had negligible effects 328 on total variance explained in both our models for both populations (variance ≤0.02).

329 Diapause incidence was higher at 17 °C 10:14 h LD than at 20 °C, 16:8 h LD, for both the 330 Canadian (76.9 ± 2.5 % vs. 9.0 ± 1.5 %, GLMM, χ²=250, df=1, p<0.001) and the French (27.9 ± 331 2.1 % vs. 0 %, GLMM, χ²=238, df=1, p<0.001) populations. At 20 °C, 16:8 h LD, low levels of 332 diapause were observed for the Canadian population, except when parasitizing oviparous aphids 333 (Fig. 2), whereas no diapause was expressed for the French population.

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335 Some female parasitoids produced offspring that had stronger responses to changes in host 336 morph or abiotic conditions than offspring of other females (Fig. 3). Data for each female are 337 made available as a supplementary material sheet. In some female’s brood, there was no 338 variation in diapause plasticity in response to different biotic (morphs) or abiotic (photoperiod 339 and temperature) conditions (RN slope = 0). In the Canadian population at 17°C 10:14 h L:D, 340 reaction norm slopes (i.e., diapause level variations between conditions within a single brood) 341 ranged from -71% to 48%, for the diapause response to either oviparous or viviparous morphs 342 Fig. 3A). At 20°C 16:8 h L:D, these RN slopes ranged from -50% to 12% (Fig. 3B). In the 343 French population at 17°C 10:14 h L:D, RN slopes ranged from -29% to 38% for the diapause 344 response to either oviparous or viviparous morphs (Fig. 3C). 345 In the Canadian population, for parasitoids developing in viviparous morphs, RN slopes 346 ranged from -100% to -3% (Fig. 3D), and for parasitoids developing in oviparous morphs, RN 347 slopes ranged from -100% to -12% (Fig. 3E), for the diapause response to abiotic conditions 348 (17°C 10:14 h L:D vs. 20°C 16:8 h L:D). In the French population, for parasitoids developing in 349 viviparous morphs, RN slopes ranged from -80% to 0% (Fig. 3F), and for parasitoids 350 developing in oviparous morphs, RN slopes ranged from -50% to 0% (Fig. 3G) for the diapause 351 response to either abiotic conditions.

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354 Figure 2: Percent diapause incidence (± SE)CI95%) in two Aphidius ervi populations. Left: Canadian population 355 naturally experiencing harsh winter. Right: French population naturally experiencing mild winter. For both 356 populations, three different morphs of the pea aphid Acyrthosiphon pisum (oviparous sexual females, viviparous 357 parthenogenetic females produced under sexual-inductive conditions),, and control viviparous females produced 358 under non- sexual-inductive conditions) were used for parasitoid development, under two abiotic conditions 359 (17 °C, 10:14 h LDL:D or 20°C, 16:8 h LDL:D). For each treatment, N represents the total number of parasitoid 360 mummies used to calculate diapause incidence.

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363 Figure 3: Reaction norms (RN) of diapause levels in the offspring of each parasitoid female from each parasitoid population (Canadian: and French: ), both within 364 morphs at different abiotic conditions (top panel, A & C: 17°C 10:14 h L:D, B: 20°C 16:8 h L:D)), and within abiotic conditions between morphs (bottom panel, D & F:

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365 oviparous morphs, E & G: viviparous morphs). RN for the French population at 20°C 16:8 h L:D are not displayed as no diapause was observed under these conditions. N=24 366 parasitoid female per condition. Note that some lines may be confounded.

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367 Metabolomic analyses and lipid reserves of aphid host morphs

368 All measured compounds were found in both aphid morphs. The first and second principal 369 component (PC1 and PC2, respectively) of the PCA, accounted for 37.1 % and 26 % of the total 370 inertia, respectively (Fig. 3). Oviparous and viviparous female hosts were separated on PC1, 371 with oviparous females exhibiting significantly higher concentrations of trehalose, ribose, 372 arabitol, GABAgamma aminobutyric acid and mannose than viviparous ones (ANOVA, df=1, 373 p<0.05) (Fig. S1). Conversely, viviparous hosts had significantly higher concentrations of 374 alanine, gluconolactone, dopamine, putrescine, phenylalanine, glycerol, proline and quinic acid 375 than oviparous aphids (ANOVA, df=1, p<0.05) (details of metabolite amounts measured from 376 each morph are provided in Fig. S1). The second component of the PCA depicted the inter- 377 individual variation of metabolites within each of the two morphs (Fig. 34).

378 The analysis by metabolic family revealed that sugars / sugar phosphates (at the exception of 379 glucose) and polyols were measured in higher amounts in oviparous morphs, while amino acids, 380 amines and other metabolites were generally found in higher concentrations in viviparous hosts 381 (Fig. S2). Altogether, metabolic differences among oviparous and viviparous females revealed 382 that activities of the pathways involved in aminoacyl-tRNA biosynthesis and glutathione 383 metabolism were higher in viviparous females.

384 Oviparous hosts had a higher fat content ratio (mg fat/mg dry mass) than viviparous ones Mis en forme : Justifié 385 (0.63 ±0.02 and 0.51 ±0.03, n= 52 and n= 23, respectively) (ANOVA, LR=8.0, df=1, p<0.005). 386 The fat mass represented 37.8 ±0.8% and 33.3 ±1.3% of the dry mass of oviparous and 387 viviparous morphs, respectively.

388

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389

390 Figure 34: Multivariate analysis (PCA) on the first two principal components (PC) representing links between 391 metabolic compounds (47 log-transformed variables, nmol.mg-1) and two aphid morphs (oviparous vs. 392 viviparous females) of Acyrthosiphon pisum. Enclosed figure in the upper panel shows a PCA of the six 393 metabolite categories. Confidence ellipses (95%) are constructed around each aphid group centroid (n=9 394 replicates by morph). Contributions of metabolite variables to PC1 and PC2 are provided in the two lower 395 panels. Abbreviations are listed in Table 1.supplementary figure S3. Abbreviations are Alanine (Ala), Aspartic 396 acid (Asp_Ac), Cadaverine (Cad), Citric acid (Cit_Ac), Citrulline (Citr), Dopamine (Dop), Fructose-6-phosphate 397 (F6P), Galacturonic acid (Gal_Ac), Gamma aminobutyric acid (GABA), Gluconolactone (GNL), Glucose-6- 398 phosphate (G6P), Glutamic acid (Glu), Glyceric acid (Glyc_Ac), Glycerol-3-phosphate (Gly3P), Glycine (Gly), 399 Isoleucine (Ile), Lactic acid (Lact_Ac), Leucine (Leu), Lysine (Lys), Malic acid (Mal_Ac), Ornithine (Orn), 400 Phenylalanine (Phe), Phosphoric acid (Phos_Ac), Pipecolic acid (Pipe_Ac), Proline (Pro), Putrescine (Put), 401 Quinic acid (Quin_Ac), Serine (Ser), Threonine (Thr), Triethanolamine (TEA), Valine (Val).

402 Mis en forme : Espace Après : 0 pt

403 Discussion

404 Species interactions greatly contribute in shaping ’ seasonal ecological strategies, 405 because one species needs to synchronize or unsynchronize its life-cycle with interacting 406 partners or antagonists. However, biotic-induced diapause signals are poorly studied. A few 407 cases of predator-induced diapause have been documented in arthropods (Ślusarczyk 1995, 408 Kroon et al. 2008)(Ślusarczyk 1995, Kroon et al. 2008), such as in Daphnia magna 409 (Diplostraca: Daphniidae) in which the production of diapausing eggs is stimulated by predator 410 exudates and chemicals originating from injured conspecifics (Ślusarczyk 1999)(Ślusarczyk 411 1999). Reversely, low prey density was reported to influence summer diapause of the lady 412 beetle Hippodamia undecimnotata (Coleoptera: Coccinellidae) (Iperti and Hodek 1974)(Iperti 413 and Hodek 1974). Also, in herbivorous insects that require strong synchrony with their host 414 plant phenology, resuming activities after winter diapause is also influenced by the 415 physiological status of the plant (Leather et al. 1993)(Leather et al. 1993). Similarly, the host 416 plays a major role in parasitoid seasonal ecology. In addition to abiotic factors, such as 417 photoperiod and temperature, the host genotype, species, size, life-stage and abundance can 418 modulate parasitoid diapause (Tauber et al. 1986, Danks 1987)(Tauber et al. 1986, Danks 1987).

419 We report that parasitoids can use host oviparous morph as a cue for diapause induction, 420 with higher diapause incidence (up to 20%) expressed in A. ervi developing in oviparous 421 A. pisum females compared to viviparous conspecifics. This pattern is likely due to differences 422 in host physiology and metabolic contents. However, we have observed relatively high

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423 intrapopulation variability within each female’s offspring in response to the host morph, and in 424 a lower extent in response to abiotic conditions, through the study of reaction norms. 425 Polymorphism in the response of diapause induction cues (i.e., in plasticity) is known to be 426 responsible for variability in diapause levels within populations experiencing different 427 environmental conditions, but is still to be more deeply explored. As expected, parasitoids from 428 the harsh winter environment expressed higher diapause levels than parasitoids from the mild 429 winter environment. Of significance, only parasitoids from the harsh winter area and exposed to 430 summer-like conditions relied on host morph as a cue for diapause induction.

431 Parasitoid populations of A. ervi from contrasted climatic environments (Canada and France) 432 do not respond the same way to abiotic (photoperiod and temperature) and host cues. The 433 French population of Aphidius spp. evolved under warming temperature conditions over the past 434 decades, and this has allowed individuals of this species to remain active under mild winter 435 conditions prevailing in this area, with none or small proportions of individuals entering 436 diapause (Tougeron et al. 2017b). In mild winter areas, non-diapausing parasitoids maintain 437 their populations by exploiting asexual anholocyclic aphid hosts during winter periods (Langer 438 and Hance 2000, Andrade et al. 2015, 2016) as sexual morphs are rare in these areas (Dedryver 439 et al. 2001). Diapause expression can be genetically lost or reduced in insects when they do not 440 experience the necessary environmental factors for its induction (e.g., Bradshaw and Holzapfel 441 2001, Gariepy et al. 2015). Consequently, parasitoid populations from mild winter areas may 442 not have evolved a response to sexual hosts, or they may have lost the capacity to answer such a 443 cue to enter diapause(Tougeron et al. 2017b). In mild winter areas, non-diapausing parasitoids 444 maintain their populations by exploiting asexual anholocyclic aphid hosts during winter periods 445 (Langer and Hance 2000, Andrade et al. 2015, 2016) as sexual morphs are rare in these areas 446 (Dedryver et al. 2001). Diapause expression can be genetically lost or reduced in insects when 447 they do not experience the necessary environmental factors for its induction (e.g., Bradshaw and 448 Holzapfel 2001, Gariepy et al. 2015). Consequently, parasitoid populations from mild winter 449 areas may not have evolved a response to sexual hosts, or they may have lost this capacity under 450 changing environments.

451 The opposite pattern is observed in Canadian populations, where all aphid parasitoids enter 452 diapause during winter (Brodeur and McNeil 1994)(Brodeur and McNeil 1994). In these cold 453 temperate regions, sexual morphs of aphids are produced at the end of the growing season, and 454 represent the last hosts available for parasitoids before the onset of unfavorable winter 455 conditions. In addition, parasitism of aphid sexual morphs on primary host plants allows 456 parasitoids to overwinter nearby their hosts, thereby favoring host availability in spring for 457 newly emerged parasitoids, and improving reproductive-cycles synchronization (Höller 1990, 458 Christiansen-Weniger and Hardie 1997)(Höller 1990, Christiansen-Weniger and Hardie 1997). 459 In regions with harsh winter climates, parasitoids have coevolved with the seasonal occurrence 460 of host morphs and may use oviparous morphs as a convergent signal with temperature and 461 photoperiod decrease in fall to enter diapause. Canadian Aphidiinae parasitoids begin to 462 overwinter as early as mid-July, with all individuals being in diapause by early September 463 (Brodeur and McNeil 1994, Tougeron et al. 2018)(Brodeur and McNeil 1994, Tougeron et al. 464 2018). This seasonal pattern might be an adaptation to avoid early lethal frosts. Moreover, we 465 showed that oviparous hosts only influenced diapause under summer-like conditions, suggesting 466 that encountering this morph informs the parasitoids for upcoming deleterious conditions and 467 modulates diapause expression. In natural settings, alternative host species can be present, and 468 both anholocyclic and holocyclic aphid populations can coexist (Dedryver et al. 2001)(Dedryver

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469 et al. 2001), which may send confounding signals to parasitoids, and may explain why only a 470 fraction of the population responded to oviparous morphs. In Canada, oviparous morphs of the 471 pea aphid are present in the environment as soon as August (Lamb and Pointing 1972)(Lamb 472 and Pointing 1972). In fall-like conditions, the morph effect was overridden by the 473 temperature/photoperiod effect, which remains the main signal for diapause induction. 474 Alternative diapause-inducing cues such as those associated with the host are usually viewed as 475 factors modulating diapause expression which is mainly triggered by temperature and 476 photoperiod (Tauber et al. 1986)(Tauber et al. 1986). For example, in the polyphagous 477 herbivore Choristoneura rosaceana (Lepidoptera: Tortricidae), diapause is dependent upon 478 photoperiod and temperature, but under similar abiotic conditions, the proportion of larvae 479 entering diapause differs depending on the host-plant species (Hunter and McNeil 480 1997).(Hunter and McNeil 1997). Moreover, the effect of the host-plant was observed even 481 under photoperiod and temperature conditions known to induce low levels of diapause (Hunter 482 and McNeil 1997)(Hunter and McNeil 1997). The relative importance of each environmental 483 cue at inducing diapause in insects remains to be evaluated for a significant number of species.

484 Parasitoids’ response to host morph could be partly shaped by maternal effects, as females 485 have the capacity to assess host quality through a combination of physiological, morphological 486 or, behavioural and chemical cues (van Baaren and Nénon 1996, Boivin et al. 2012)(van Baaren 487 and Nénon 1996, Boivin et al. 2012). Developing immature parasitoids may also directly 488 respond to the quality and quantity of metabolites available from hosts, which could trigger the 489 onset of diapause. The overwintering metabolic and physiological syndrome is highly conserved 490 among insects (Tauber et al. 1986)(Tauber et al. 1986), and both hosts and parasitoids may 491 respond to the same molecules involved in diapause initiation. As an example concurring to this 492 hypothesis, diapausing prepupae of the aphid parasitoid Praon volucre (Hymenoptera: 493 Braconidae) showed similar proportions of some sugars (e.g. trehalose, fructose) and polyols 494 (e.g. arabitol) (Colinet et al. 2012)(Colinet et al. 2012) than non-parasitized oviparous morphs 495 of the pea aphid tested in our study. Our results suggest that high concentrations of some 496 polyols and sugar metabolites in the oviparous morphs, as well as accumulation of fat reserves 497 associated with the overwintering process, may either directly contribute to induce diapause in 498 parasitoids developing in such hosts or may trigger the internal physiological cascade 499 responsible for parasitoid diapause.

500 In the present work, oviparous A. pisum females have higher fat reserves than their 501 viviparous counterparts. This finding is consistent with the metabolic phenotypes of the hosts, 502 which revealed higher levels of sugar and sugar phosphate metabolites from the glycolytic 503 pathway in oviparous females, this pathway providing elementary bricks for fatty acid and 504 triacylglyceride (TAG) synthesis. Fatty acids serve as a main source of energy for physiological 505 or ecological processes, including flight, gametes production, egg maturation and hormones 506 synthesis (Arrese and Soulages 2010), and have been shown to represent up to 30% of aphids’ 507 fresh mass (Dillwith et al. 1993, Sayah 2008). Interestingly, lipids can provide energy for 508 overwintering insects and sugars can be metabolized to produce sugar-based cryoprotectant 509 molecules (Storey and Storey 1991, Hahn and Denlinger 2011, Sinclair and Marshall 2018). In 510 oviparous females, the need for TAG may be higher than in viviparous ones, as eggs with yolk 511 (vitellus) are mostly composed of fat and proteins (Brough and Dixon 1990). Also, reserves 512 from the fat-body, including TAG and glycogen, play major roles in overwintering insects, 513 including diapause (reviewed in Sinclair and Marshall 2018) and could explain why oviparous 514 aphids have high fat content to prepare their eggs for successful overwintering. Diapause entails

17

515 important energetic costs for insects (Ellers and Van Alphen 2002, Hahn and Denlinger 2011) 516 and they may enter diapause only when a critical body-mass or amount of energetic reserves has 517 been reached (Colinet et al. 2010); for parasitoids, developing in an oviparous host could 518 contribute to reach this level.

519 Metabolites acting as compatible solutes greatly contribute to cold hardiness and 520 overwintering survival (Storey and Storey 1991, Bale 2002, Hodkova and Hodek 2004).(Storey 521 and Storey 1991, Bale 2002, Hodkova and Hodek 2004). Metabolic analyses identified sugars 522 and polyols in higher amounts in oviparous females containing eggs intended to overwinter. 523 Overwintering eggs of the aphid Hyalopterus pruni (Homoptera: Aphididae) are characterized 524 by high values of mannitol and trehalose (Sömme 1969)(Sömme 1969), as also observed in our 525 A. pisum oviparous morphs. Glucose-6-phosphate and fructose were found at high 526 concentrations in oviparous morphs of A. pisum and are precursors of sorbitol (Storey and 527 Storey 1991)(Storey and Storey 1991), a cryoprotective compound also observed in diapausing 528 individuals of P. volucre parasitoids (Colinet et al. 2012).(Colinet et al. 2012). Fructose-6- 529 phosphate is a precursor of mannitol, and both are cryoprotectant molecules (Storey and Storey 530 1991)(Storey and Storey 1991) highly concentrated in oviparous female hosts, and found in 531 most of overwintering insects (Leather et al. 1993)(Leather et al. 1993). These metabolites may 532 be responsible for diapause induction in parasitoids developing in oviparous morphs. 533 GABAGamma aminobutyric acid was more concentrated in oviparous females and could also 534 serve as an indirect seasonal cue for parasitoids because this neurotransmitter is known to be 535 involved in insect perception of photoperiodic changes (Vieira et al. 2005)(Vieira et al. 2005).

536 Surprisingly, in viviparous females, we found high concentrations of glycerol a 537 cryoprotective compound usually associated with the diapause syndrome (Hayward et al. 538 2005).(Hayward et al. 2005). As suggested by the high concentrations of glucose observed in 539 these females, glycogen production through gluconeogenesis pathway could be used as main 540 source of energy by these viviparous morphs (Dixon 1985)(Dixon 1985). In addition, observed 541 physiological differences between host morphs are not necessarily linked to overwintering 542 strategies. For example, viviparous aphids have high concentrations of proline, which is used as 543 fuel for insect flight (Teulier et al. 2016).(Teulier et al. 2016). Viviparous aphids can rapidly 544 produce alatewinged individuals for dispersal in case of overcrowding or degradation of host 545 plant quality (Hardie 1980)(Hardie 1980).

546 To conclude, intra- and interspecific interactions are of primary importance for ecosystem 547 functions, such as biological control, but still require deeper investigations in the context of 548 diapause and seasonal strategies. Overwintering strategies are rapidly shifting in the context of 549 climate change (Bradshaw and Holzapfel 2001, Bale and Hayward 2010) and may cause 550 temporal mismatches between trophically interacting species (Tylianakis et al. 2008, Walther 551 2010). Thus, potential bottom-up effects on diapause, such as reported in our study, should be 552 given more attention and should be considered as a potential factor explaining the low levels of 553 diapause expression in insects from mild winter areas, together with global warming (Jeffs and 554 Lewis 2013, Andrade et al. 2016, Tougeron et al. 2017b). In addition, there was variation for 555 plasticity in diapause induction among female genotypes, mostly in response to the parasitized 556 morph but also to abiotic conditions, as determined by slopes of the reaction norms. This means 557 that there is genetic polymorphism in diapause plasticity within populations, which may allow 558 natural selection to act in the context of rapid environmental and climate changes (Sgrò et al. 559 2016). Moreover, our results are of significance for the manipulation of insect diapause; e.g., in Mis en forme : Anglais (États Unis) 560 the context of mass rearing for the food industry, or for the biological control industry. More

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561 generally, a better appreciation of the processes governing phenology is needed to predict the 562 consequences of such phenology changes on species interactions and synchrony across multiple 563 trophic levels, community functioning and ecosystem services.

564

565 Authors’ Contributions

566 KT performed the diapause experiments, analyzed the data and wrote the manuscript. KT 567 and DR performed the metabolomic experiments and analyzed the metabolomic data. All co- 568 authors substantially contributed at designing protocols and revising the manuscript.

569

570 Acknowledgments

571 We are grateful to G.both reviewers and both recommenders from PCI Ecology who made 572 an excellent job in reviewing our manuscript and in providing strong advices on how to improve 573 it. We thank G. Le Trionnaire at INRA Le Rheu for providing the aphid clones. We thank S. 574 Llopis and J. Doyon for technical support and J. Jaquiéry for stimulating discussions. KT was 575 funded by the Fyssen foundation, by the French Région Bretagne (ARED grant) and by the 576 Canada Research Chair in Biological Control awarded to JB.

577

578 Data accessibility

579 Metabolomics and diapause data have been made publicly available as a supplementary 580 material attached to this publication.

581

582 References

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713 Polgár, L. A., and J. Hardie. 2000. Diapause induction in aphid parasitoids. Entomologia 714 Experimentalis et Applicata 97:21–27. 715 Polgár, L. A., M. Mackauer, and W. Völkl. 1991. Diapause induction in two species of aphid 716 parasitoids: the influence of aphid morph. Journal of insect physiology 37:699–702. 717 R Core Team. 2017. R: A language and environment for statistical computing. R Foundation for 718 Statistical Computing, Vienna, Austria. 719 Sayah, F. 2008. Changes in the lipid and fatty acid composition of hemolymph and ovaries during the 720 reproductive cycle of Labidura riparia. Entomological Science 11:55–63. Mis en forme : Police :Italique 721 Sgrò, C. M., J. S. Terblanche, and A. A. Hoffmann. 2016. What Can Plasticity Contribute to Insect 722 Responses to Climate Change? Annual Review of Entomology 61:433–451. 723 Sinclair, B. J., and K. E. Marshall. 2018. The many roles of fats in overwintering insects. The Journal Mis en forme : Espace Avant : 0 pt 724 of Experimental Biology 221:jeb161836. 725 Ślusarczyk, M. 1995. Predator-Induced Diapause in Daphnia. Ecology 76:1008–1013. Mis en forme : Police :Italique 726 Ślusarczyk, M. 1999. Predator-induced diapause in Daphnia magna may require two chemical cues. Mis en forme : Police :Italique 727 Oecologia 119:159–165. 728 Sömme, L. 1969. Mannitol and glycerol in overwintering aphid eggs. Norsk Entomologisk Tidsskrift 729 16:107–111. 730 Storey, K. B., and J. M. Storey. 1991. Biochemistry of cryoprotectants. Pages 64–93 Insects at low Mis en forme : Police :Italique 731 temperature. Pages 64-93. Lee R. (ed.). Springer, The Netherlands. 732 Tauber, M. J., C. A. Tauber, and S. Masaki. 1986. Seasonal adaptations of insects. Oxford university 733 press, New-York, USA. 734 Terblanche, J. ., C. . Klok, and S. . Chown. 2004. Metabolic rate variation in Glossina pallidipes Mis en forme : Police :Italique 735 (Diptera: Glossinidae): gender, ageing and repeatability. Journal of Insect Physiology 50:419– 736 428. 737 Teulier, L., J.-M. Weber, J. Crevier, and C.-A. Darveau. 2016. Proline as a fuel for insect flight: 738 enhancing carbohydrate oxidation in hymenopterans. Proceedings of the Royal Society B: 739 Biological Sciences 283:20160333. 740 Thompson, J. N. 1988. Variation in interspecific interactions. Annual review of ecology and 741 systematics 19:65–87. 742 Tougeron, K., G. Hraoui, C. Le Lann, J. Van Baaren, and J. Brodeur. 2017a. Intraspecific maternal 743 competition induces summer diapause in insect parasitoids. Insect Science 25:1080–1088. 744 Tougeron, K., C. Le Lann, J. Brodeur, and J. van Baaren. 2017b. Are aphid parasitoids from mild 745 winter climates losing their winter diapause? Oecologia 183:619–629. 746 Tougeron, K., J. Van Baaren, S. Llopis, A. Ridel, J. Doyon, J. Brodeur, and C. Le Lann. 2018. 747 Disentangling plasticity from local adaptationsadaptation in diapause expression of 748 parasitoidsin parasitoid wasps from and within contrastedcontrasting thermal environments.: a 749 reciprocal translocation experiment. Biological Journal of the Linnean Society 124:756–764. 750 Tylianakis, J. M., R. K. Didham, J. Bascompte, and D. A. Wardle. 2008. Global change and species 751 interactions in terrestrial ecosystems. Ecology Letters 11:1351–1363. 752 Vieira, R., J. M. Míguez, and M. Aldegunde. 2005. GABA modulates day–night variation in 753 melatonin levels in the cerebral ganglia of the damselfly Ischnura graellsii and the Mis en forme : Police :Italique 754 grasshopper Oedipoda caerulescens. Neuroscience letters 376:111–115. Mis en forme : Police :Italique 755 Walther, G.-R. 2010. Community and ecosystem responses to recent climate change. Philosophical 756 Transactions of the Royal Society B: Biological Sciences 365:2019–2024.

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